Bottom Line:
Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively.Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers.These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

ABSTRACTCirculating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

Mentions:
To evaluate the performance (sensitivity and specificity) of this 3D Pd filter device in metastatic disease in vivo, we developed mouse CTC models as reported previously [34], [35] and tested the diagnostic potential of this device. GFP-expressing metastatic colonic cancer cells (COLM5-EGFP) were subcutaneously (sc) injected, and 2–3 months later, they metastasized to the lung spontaneously. Lung metastasis at the micrometastasis level was observed 1–2 months after sc injection of metastatic tumor cells (Figures 5A–5D) and macroscopic lung metastasis was observed 2–3 months post sc injection (Figures 5E–5I). Using this metastasis model, we tested whether or not CTC can be detected by this device. In this spontaneous mouse metastasis model, we could reproducibly detect a significant number of CTCs (10–1000<) in the blood of mice 2–3 months after sc injection (Figure 5L and 5M), but not in mice 1–2 months after injection (Figure 5J and 5K). The number of CTCs clearly increased with progression of lung metastasis (Figure 5N), but the lung metastasis still remained small at less than 1 mm in diameter in this CTC model (Figure 5F), indicating that CTC appears at a relatively early stage of metastatic development in mice.

Mentions:
To evaluate the performance (sensitivity and specificity) of this 3D Pd filter device in metastatic disease in vivo, we developed mouse CTC models as reported previously [34], [35] and tested the diagnostic potential of this device. GFP-expressing metastatic colonic cancer cells (COLM5-EGFP) were subcutaneously (sc) injected, and 2–3 months later, they metastasized to the lung spontaneously. Lung metastasis at the micrometastasis level was observed 1–2 months after sc injection of metastatic tumor cells (Figures 5A–5D) and macroscopic lung metastasis was observed 2–3 months post sc injection (Figures 5E–5I). Using this metastasis model, we tested whether or not CTC can be detected by this device. In this spontaneous mouse metastasis model, we could reproducibly detect a significant number of CTCs (10–1000<) in the blood of mice 2–3 months after sc injection (Figure 5L and 5M), but not in mice 1–2 months after injection (Figure 5J and 5K). The number of CTCs clearly increased with progression of lung metastasis (Figure 5N), but the lung metastasis still remained small at less than 1 mm in diameter in this CTC model (Figure 5F), indicating that CTC appears at a relatively early stage of metastatic development in mice.

Bottom Line:
Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively.Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers.These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.

ABSTRACTCirculating tumor cells (CTCs) in the blood of patients with epithelial malignancies provide a promising and minimally invasive source for early detection of metastasis, monitoring of therapeutic effects and basic research addressing the mechanism of metastasis. In this study, we developed a new filtration-based, sensitive CTC isolation device. This device consists of a 3-dimensional (3D) palladium (Pd) filter with an 8 µm-sized pore in the lower layer and a 30 µm-sized pocket in the upper layer to trap CTCs on a filter micro-fabricated by precise lithography plus electroforming process. This is a simple pump-less device driven by gravity flow and can enrich CTCs from whole blood within 20 min. After on-device staining of CTCs for 30 min, the filter cassette was removed from the device, fixed in a cassette holder and set up on the upright fluorescence microscope. Enumeration and isolation of CTCs for subsequent genetic analysis from the beginning were completed within 1.5 hr and 2 hr, respectively. Cell spike experiments demonstrated that the recovery rate of tumor cells from blood by this Pd filter device was more than 85%. Single living tumor cells were efficiently isolated from these spiked tumor cells by a micromanipulator, and KRAS mutation, HER2 gene amplification and overexpression, for example, were successfully detected from such isolated single tumor cells. Sequential analysis of blood from mice bearing metastasis revealed that CTC increased with progression of metastasis. Furthermore, a significant increase in the number of CTCs from the blood of patients with metastatic breast cancer was observed compared with patients without metastasis and healthy volunteers. These results suggest that this new 3D Pd filter-based device would be a useful tool for the rapid, cost effective and sensitive detection, enumeration, isolation and genetic analysis of CTCs from peripheral blood in both preclinical and clinical settings.